1. Field of the Invention
The present invention relates to a fungal antigen effective for infectious diseases caused by fungi, which are pathogenic microorganisms, having cell wall, for prevention or treatment of allergoses, and for diagnosis of diseases caused by fungi, and a process for producing the same.
2. Discussion of the Related Art
It has been known that fungi infect vertebrates such as humans and animals to cause all kinds of diseases. For example, superficial mycosis is caused in human skin, oral, or the like; systemic mycosis is caused in internal organs, brain, or the like, and similar infectious diseases are also caused to animals such as pets and domestic animals. Among them, Candida, such as Candida albicans, Cryptococcus, such as Cryptococcus neoformans, Aspergillus, such as Aspergillus fumigatus, Pneumocystis carinii, or the like have been known as major causative fungi which cause systemic mycosis by infecting humans. Candida which infects skin, oral, vagina, or the like, and Dermatophytes such as Trichophyton mentagrophytes and Trichophyton rubrum which infects skin of hands, feet, or the like have been taken for major causative fungi for superficial mycosis.
A lot of Dermophytes are fungi which cause infectious diseases to domestic animals, and the like, and it has been known that Microsporum such as Microsporum canis and Microsporum gypseum are such fungi other than Trichophyton, such as Trichophyton verrucosum mentioned above. In addition to these fungi, a wide variety of fungi occur in the living environment, and are assumed to infect humans and animals. Furthermore, recently, by the frequent use of a wide range of antibiotics, use of immunosuppressants, use of immunosuppressive anticancer agents, etc., patients administered with these drugs have become immunocompromised hosts, and opportunistic infection with fungi of low pathogenicity have been increased in normal individuals administered therewith. Also, AIDS patients suffer from frequent onset of thrush and complications of various mycoses. Patients on treatment with intravascular catheter indwelling, especially intravenous hyperalimentation (IVH), are likely to develop infectious diseases caused by fungi, especially with Candida owing to catheter.
On the other hand, allergoses, typically including asthma, atopic dermatitis, and allergic rhinitis, have been increasing dramatically, among which a very large number of allergoses are caused by fungi.
As for a lot of allergoses, because of sensitization with a causative antigen of its disease, an IgE antibody (reagin antibody) specific to the antigen as an allergen is produced in serum and tissue, so that the IgE antibody is bound to mast cells and basophil receptors. When re-exposed to the same antigen, the IgE bound to the cells is crosslinked with the antigen on the cell surface, thereby resulting in physiological effects of IgE-antigen interaction. These physiological effects are exhibited via a release of chemical mediators, such as histamine, serotonin, heparin, eosinophilic chemotactic factor, and various leukotrienes. These effects can be systemic or topical, depending on the route of an antigen entering the body and the pattern of IgE sedimentation on the mast cells or basophils.
The systemic symptoms include anaphylactic shock, which causes intravascular IgE-basophil response to the antigen. As a consequence, smooth muscle contraction and capillary dilation take place as major changes, thereby resulting in symptoms such as eruption, vomiting, diarrhea, and dyspnea. In more severe cases, it may lead to death. In addition, the topical symptoms generally develop on the epithelium surface at the site of an antigen entering the body as shown by reddening and papules. When bronchiolar smooth muscle contraction develops as a topical symptom, it is manifested as bronchial asthma.
As the causative strains for causing allergoses, there have been known Penicillium, Candida, Aspergillus, Alternaria, Cladosporium, Malassezia, Botrytis, Mucor, Rhizopus, Aureobasidium, Fusarium, Trichoderma, Helminthosporium, Neurospora, Wallemia, Rhodotorula, and Trichophyton. 
As the therapy for fungal infections, a treatment with an antifungal agent is generally employed. A large number of drugs for superficial mycoses have been developed, and some excellent drugs for systemic infections are available. In terms of efficacy, toxicity, adverse reactions, etc., however, their effects are unsatisfactory. For example, amphotericin B that has long been used, causes various adverse reactions, including serious renal dysfunction. Although various azole antifungal agents, typically including fluconazole, have been developed, infections are highly likely to recur because their action is static. Also, resistant strains are emerging due to frequent use. As the resistant strains emerge, the cross-resistance takes place, because many of the antifungal agents presently in practical use possess similar action mechanisms, which can pose a major problem. In cases of superficial mycoses, various therapeutic drugs have been developed, but none can be said to be satisfactory, because it requires a long-term treatment period and recurrence is repeated. Therefore, a development of a further improved drug has been in demand. Moreover, since a treatment with topical preparations only would be unsatisfactory for some superficial mycoses, e.g., nail tinea, these superficial mycoses would require systemic medication such as griseofulvin. In this case, long-term administration would be necessitated, which can cause various adverse reactions by the drugs. Also, as in superficial mycoses and AIDS-related thrush, since repetitive infection is caused, there is a major problem in terms of costs, even if an effective antifungal agent is developed. As described above, a treatment with an antifungal agent has various problems.
The living body naturally possesses an ability to protect against infection by fighting against such foreign-invading microorganisms. Vaccines utilize this ability. The prevention against infection with pathogenic bacteria has been carried out by vaccines and has been long used with fair efficacy. For such vaccines against bacterial infectious diseases, attenuated bacteria (Mycobacterium tuberculosis), killed bacteria (Vibrio cholerae), toxoids (Corynebacterium diphtheriae, Clostridium tetani), or purified antigens from capsular polysaccharides on cell surface (Bordetella pertussis, Streptococcus pneumoniae, influenza virus, Neisseria meningitidis) are employed as antigens. The vaccines provide an ability to protect against infection to the host by antibodies against antigenic molecules of the pathogen and by cellular immunity. It is considered that the antibodies serve to neutralize the toxic substances secreted by pathogens, and to prevent pathogens from invading host cells by binding to the cell surface molecules of the pathogen. In the cellular immunity, CD4+ cells and CD8+ T cells play a key role for recognizing the antigenic molecules of the pathogen and activating a protection reaction specific to the pathogen. Immunogenic substances, which are antigenic molecules possessed by the pathogens, have been isolated and identified, and some studies using these immunogens as sensitizing antigens (vaccines) have been made. In such cases, capsular polysaccharides, which are cell surface molecules as described above, are commonly used as immunogens.
An extremely large number and many kinds of fungi are present in the environment, and almost all vertebrates are sensitized with these fungi. Also, a large number of fungi are commonly present in the living bodies. The vertebrates are, therefore, generally provided with various immunological reactions for body protection against these fungi. Immunological reactions which have important roles against fungal infections show the phagocytosis and fungicidal actions of activated macrophages and polymorphonuclear leukocytes (PMN) and play a main role, and are also known to contribute to antibodies and cellular immunity. On their cell surface, fungi have a cell wall, comprising, as a main component, polysaccharides, such as mannan, glucan, and chitin, of which the content accounts for nearly 30% of the entire cell in some fungal cells [Klis, R. U. et al., Yeast, Vol. 10, 851-869, (1994)]. Of these cell wall components, mannan is the most antigenic. The mannan is a polysaccharide in the cell surface layer, and an antibody against the polysaccharide moiety is produced in large amounts. The cell wall glucans from fungi, typically including Zymosan, possess various biological activities, and are known to possess non-specific immunopotentiating actions. It is assumed that the cell wall components, including mannan on a cell surface of fungi, play an important role in causing infection as an adhesion molecule to the living body of cells.
Also, Cryptococcus galactoxylomannan [Devi, S. J. N. et al., Infect. Immun., Vol. 59, 3700-3707 (1991)] and the Candida albicans adhesion factor phosphomannoprotein (WO 95/31998) have been reported to serve as vaccines, and antibodies against these antigenic molecules have been reported to possess protection activity against infection. Regarding the induction of immunological protection against infection with living or dead Candida cells, a large number of reports have been made [Segal, E. et al., Critical Reviews in Microbiology, Vol. 14, 229-271 (1987)]. In this case as well, it has been assumed that an immunological reaction mainly functions for body protection against the cell wall components which are the cell surface molecules.
Other vaccines against fungi include the ribosome vaccine [Segal, E., Handbook of Applied Mycology, Volume 2: Immunizations against fungal diseases in man and animals., Humans, animals and insects] has been tested for infectious diseases caused by fungi, typically including Candida albicans and Trichophyton, and studied on laboratory animals and partially on humans and domestic animals. Recently, there have been reported that enolase and stress protein HSP90 (Japanese Unexamined Patent Publication No. Hei 4-502257) can induce protective activity against infection.
However, it cannot be said that all of the above-mentioned antigenic molecules are confirmed to have satisfactory efficacy. Also, it is doubtful whether or not satisfactory efficacy can be obtained in highly diversified mammals by treatment with a single antigenic molecule.
On the other hand, therapies for allergoses include the use of antihistaminic drugs, steroidal anti-inflammatory drugs, chemical mediator release suppressors, and the like. It should be noted, however, that the antihistamines have a risk of developing various adverse reactions, such as malaise, drowsiness, and vertigo, that the steroids have a risk of developing various adverse reactions, such as adrenal atrophy and dysfunction, and gastric ulcer, and that the chemical mediator release suppressors have a risk of also suppressing the action of chemical mediators involved in conditions other than the allergosis of interest. From this viewpoint, prevention method for reducing the chance of exposure to allergens specified by antigen diagnosis, and/or desensitization therapy using such causative allergens is considered to be an excellent therapy.
In allergoses, it is therefore necessary to first diagnose for identifying the causative antigen, and for this purpose, more than 100 kinds of commercially available allergen extracts, sometimes those prepared by the laboratory, are subjected to intradermal test for suspected antigen extracts. After a highly likely antigen is found, the antigen can be specified by determination of IgE antibody titer in sera, challenge test, or histamine release test using whole blood or lymphocytes.
As allergens by which allergic symptoms are provoked in humans, a large number of naturally occurring ones have been known. Commercially available food and other allergen extracts are supplied as crude extracts from natural allergens. Therefore, they are naturally agglomerates of many substances and contain a plurality of antigens. Recently, as a result of advances in separation and purification techniques and evaluation methods for allergen activity, antigenic proteins, which comprise the main body of allergens, are isolated and identified from a variety of food allergens.
Also, from each of allergens occurring in the environment, such as mites, Cryptomeria japonica pollen, and feline hair, antigenic proteins named as Der p I [Smith, W. A. et al., Clin. Exp. Allergy, Vol. 24, 220-228 (1994)], Cry j I [Sone, T. et al., Biochem. Biophys. Res. Commun., Vol. 199, 619-625 (1994)], and Fel d I [Morgenstern, J. P. et al., Proc. Natl. Acad. Sci. USA, Vol. 88, 9690-9694 (1991)] have been isolated as major allergens. Furthermore, the genes encoding these allergen proteins have been isolated, so that pure allergen proteins can be prepared in large amounts by genetic engineering techniques.
In the meantime, efforts have been made to isolate allergens derived from fungi. Antigenic proteins have been isolated and identified from proteins existing in fungal cells. For example, alcohol dehydrogenase (Can a I) [Shen, H. D. et al., Clin. Exp. Allergy, Vol. 21, 675-681 (1991)] and enolase [Ishiguro, A. et al., Infect. Immun., Vol. 60, 1550-1557 (1992)] have been isolated from Candida albicans and identified, and ribotoxin (Asp f Ia) [Mosor, M. et al., J. Immunol., Vol. 149, 454-460 (1992)] have been isolated from Aspergillus fumigatus and identified, some of which have been known to act as allergens.
Generally, in the case of allergens from fungi, including Candida and Aspergillus, however, there are few cases where a single major allergen exists as an antigenic protein, but a plurality of antigenic proteins exist [Stewart, G. A. et al., Clin. Exp. Allergy, Vol. 26, 1020-1044 (1996)], in which different antigens by depending upon individuals, or a plurality of antigens for each individual, are recognized as allergens, to which the individuals react. In other words, even when the individuals are allergic to Candida, for instance, it is known that in many cases antigens to which each individual reacts are different antigens, and that each individual reacts to a plurality of antigens derived from Candida. 
Presently commercially available diagnostic or therapeutic allergen extracts are for the most part simple extracts or hardly purified crude extracts, so that the included ingredients are substantially uncontrolled. The allergen extracts from fungi include those from Candida, Aspergillus, Alternaria, Cladosporium, Malassezia, Penicillium, and the like. However, the methods for production thereof differ from those for the allergen extracts from naturally occurring allergens in food or the environment described above. In other words, these extracts are not supplied as cultured cells of the causative fungus per se, but prepared from an extracellular product secreted in the culture broth as raw material, which can be considered as a side-product, obtained by subjecting a representative strain belonging to each genus to a long-term cultivation in a chemically defined medium containing a limited nutrient source. Therefore, the antigen obtainable by such production method is an autolysate of cells or an extracellular secretion, which presumably comprises, as a main component, cell wall polysaccharides typically including mannan and glucan. However, neither the contents of these antigens nor the kinds of other antigenic proteins have yet been clarified. In addition, sufficient care should be paid for its use, since their quality is diversified among manufacturers.
Cell wall polysaccharides richly contained in commercially available allergen extracts from fungi, especially mannan, serve as major allergens in some patients with allergy on one hand, and even normal individuals have large amounts of IgG and IgM against cell wall polysaccharides. In addition, mannan per se, especially neutral mannan, has been known to possess toxicity, including lethal action to the mouse [Japanese Journal of Medical Mycology, Vol. 36, 203-208 (1995)]. It has been also known that cell wall glucan possesses pathological actions, including induction of inflammation [Kogan, G. et al., Biomedical and Biotechnological Advances in Industrial Polysaccharides, 251-258 (1989)].
The use of mannan and other cell wall components, which are antigens, or fungal cells per se, as vaccines, therefore, involves risks, such as causation of hypersensitivity. Also, in desensitization therapy etc. for allergoses, cell wall components do not always act as major allergens; therefore, when an allergen composition containing a cell wall component is used, its antigenicity is of concern, necessitating to be cautious when administering to humans. In this respect, presently available allergen extracts from fungi are completely unsatisfactory. Moreover, there are no known diagnostic and/or therapeutic pharmaceutical compositions in which a sufficient amount of an effective antigen is contained.
As described above, a development of novel therapeutic drugs of high efficacy and higher safety for mycoses is strongly desired, from the viewpoints of increasing incidence of mycoses, and further problems related to adverse reactions, development of resistant strains, medical costs, etc. in antifungal agents presently in use. The vaccines are advantageous over antifungal agents in many aspects, and if vaccines for such infectious diseases caused by fungi could be found, it would not only make it possible to prevent pain and weakening owing to being taken these infectious diseases, but also enable definite reduction of the dosage of drugs intended for the treatment of these infectious diseases. Furthermore, by avoiding the use of the drugs in such a way, selective pressure on pathogenic microorganisms due to overdoses of the antifungal agents is reduced, so that the prevalence of the resistant strains can be reduced. At present, however, no such highly effective vaccines have yet been found. Also, it is expected that sensitizing with a plurality of antigens has better induction of prevention against infection than sensitizing with a single antigen in the aspects of resistance and efficacy.
On the other hand, with the increase in the incidence of allergoses, numerous therapeutic or diagnostic allergen extracts have become commercially available, many of which effective ingredients, however, have not yet been clarified. As for fungi, although it remains unknown from which portions of the fungal cells the components are derived, from the methods for their production, it is assumed that its major component is polysaccharides derived from cell wall, clearly having a low content of antigenic components derived from intracellular components, and thus having a very uneven distribution in the antigenic component. For this reason, it is considered that satisfactory treatment or diagnosis cannot be carried out by using commercially available allergen extracts from fungi, and antigen extracts obtained by similar methods. Therefore, it is expected that allergen extracts having ingredients differing from those contained in conventional allergen extracts, and that the amounts of ingredients of those allergen extracts differing from those of conventional allergens exhibit high efficacy. Also, as for the present therapy of desensitization, which is considered effective for allergoses, it is necessary that an antigenic liquid is administered intradermally in small dosages at a time, once or twice a week, with increased dosage to a level maintained over a 3- to 4-month period, the administration of which is continued for 1 to 3 additional years. By the use of an antigen composition capable of easy volume increase and/or increased dosage, therefore, it is expected that an excellent therapeutic effect can be more easily obtained. Also, mammals typically including humans are generally diverse, and it is very likely that those recognized as antigens are different even if infected with, or becomes allergic to, one kind of fungus. Antigens containing sufficient amounts of diversified antigenic components are, therefore, desirable.
Furthermore, it is diagnostically important to specify the causative antigen when choosing an effective therapy, whereby highly effective and safer treatments, such as desensitization therapy using the antigen, can be carried out. It is, therefore, preferable from these viewpoints to specify unknown antigens.
Accordingly, an object of the present invention is to provide a fungal antigen that can be used for effective, safer biologic products against diseases caused by such fungi, including, for instance, vaccine compositions, compositions for desensitization therapy, and diagnostic compositions. A further object of the present invention is to provide a method for producing the fungal antigen, and a nucleic acid encoding the fungal antigen.
These and other objects of the present invention will be apparent from the following description.